The invention relates to biocompatible substrates having a surface topography which has a low tendency for cells to adhere thereto. This has particular application for the production or prostheses or implants requiring low adhesion of cells.
Many cell types have been shown to respond by shape change, and orientation to shapes such as fibres, grooves, pits and ridges; and by accelerated movement.
However, little is known about the underlying mechanisms behind this widespread reaction of cells. Explanations of contact guidance have tended to proceed along a coherent line of thought, advancing through three successive stages. First, recognising that cells will not bend or extend their microfilaments across topography that offers the cells relatively sharp angles (greater than about 10xc2x0). Secondly, through the realisation that this is a probabilistic phenomenon to the appreciation that cells develop cytoskeletal polymerisation where cells contact discontinuities in the substratum. These ideas suggest that alignment of cells is a reaction of cytoskeleton to topography. An alternative idea is that the adhesion points of the cell, the focal contacts, are prepositioned. None of these ideas explain the nature of the interaction, presumably a signalling event, between substratum and focal contact and/or cytoskeleton.
Since the reactivity of cells to topographic stimuli changes subtly with the exact dimensions of the substrate it was of interest to discover how small a feature would lead to reactions in cells. It has been shown that cells react to linear features with widths as narrow as 130 nm but clearly it is of interest whether smaller scale topography can be sensed by cells.
Grooves 5 microns wide but only 3 nm high caused a marked reaction in macrophage cells. A preliminary report was published by Wojciak-Stothard e a., Experimental Cell Research 223, 426-435 (1996).
Generally speaking, the present invention is based on the surprising observation that surfaces having a pattern of tiny projections, usually of nanometer dimensions, inhibit the adhesion of cells thereto. This has application in the preparation of surfaces which resist the adhesion of cells.
A particular aspect of the present invention provides a biocompatible substrate having a surface comprising an array of projections or pits, said projections being of a size and spacing such that cells have a low tendency to attach to the surface.
It is envisaged that the substrate will be introduced into a living or other biologically sensitive environment and is therefore to be formed of a biocompatible material. Biocompatible materials (including but not restricted to biodegradable and bioabsorbable materials) are well known in the art and will be chosen having regard to the particular biological environment into which they are to be introduced.
It is known that certain surface features promote cell adhesion thereto and can preferentially orient cell growth, particularly along the directions of grooves and ridges of micrometer dimensions. On the other hand, the present invention allows surfaces to be provided which have a reduced tendency of cell adhesion thereto. In this way, the tendency of cells to adhere or not to a particular surface can be controlled. The process can also be used to build complex tissue structures with seeding of one cell type into gaps in another cell type. For example, where the substrate includes areas of projections/pits and planar areas, cells can be preferentially adhered to the planar areas. An adhesive material (e.g. laminin) may then be coated thereon, and further cells of a different type applied thereto. The further cells adhere preferentially to the laminin-coated areas having projections/pits and avoid adhering to the pre-existing monolayers of cells (over the planar areas). Generally, the low adhesion tendency is less than 50%, particularly less than 30% of that of a corresponding planar surface. Biocompatible substrate on which it may be desirable to inhibit cell adhesion include implants, grafts and prostheses introduced into the vascular system, gastrointestinal tract, lung alveolae, synovia, connective tissue and in the eye or gum and in any part of the body which should normally have void spaces filled with liquid or gas but does notxe2x80x94as a consequence of injury or disease. A particular application is to prevent adhesion of undesired cells to ligaments under repair. For example, a biocompatible substrate for ligament repair may be patterned on an inner surface intended to be wrapped around the ligament with a surface topography intended to promote repair of the damaged ligament (see our patent application PCT/GB95/00350); and may be patterned with a surface topography on the outer side intended to prevent undesired adhesion of other types of cells to the outside of the ligament which would reduce its mobility. In other applications, a sheet-like substrate may be patterned on both sides with adhesion-reducing projections or pits, which may be used as a separation layer to prevent two adjacent structures adhering to each other.
According to the present invention, it has surprisingly been found that projections or pits of nanometer dimensions can inhibit cell adhesion. Generally, the cross sectional dimension and height of each projection is in the range 10-250 nm, particularly in the range 20-100 nm and especially 25-75 nm. Corresponding dimensions apply to pits. A projection is characterised by having an area of reduced height all around it. A pit has an area of increased height all around it. The projection or pit may be of any cross-section, including circular, oval, square, rectangular, polygonal etc.
The projections or pits will be present in an array, which may be in any chosen pattern, such as a square pattern, a rectangular pattern, a circular pattern, a rosette pattern, a random pattern etc. As will be demonstrated hereafter, the center-to-center spacing of the projections or pits can be a determinative factor in controlling the degree of resistance to cell adhesion, with wider spacings favouring reduced cell adhesion. The spacing is generally in the region 20-500 nm, particularly 40-300 nm, especially 75-200 nm.
The projections or pits will be present in an array, which may be in any chosen pattern, such as a square pattern, a rectangular pattern, a circular pattern, a rosette pattern, a random pattern etc. As will be demonstrated hereafter, the centre-to-centre spacing of the projections or pits can be a determinative factor in controlling the degree of resistance to cell adhesion, with wider spacings favouring reduced cell adhesion. The spacing is generally in the region 20-500 nm, particularly 40-300 nm, especially 75-200 nm.
The biocompatible substrate may, if desired, be formed of a resorbable material which becomes resorbed into the living tissue within a chosen period of time, usually a number of days, Following implantation. For example, a substrate intended to prevent adhesion of tissue structures during a healing process could become resorbed after fulfilling its function. Often the need to prevent such undesired cell adhesion only occurs for a limited time after injury or surgical intervention, so in many cases a short-lived form of treatment is appropriate. Bioresorbable materials are well known in the art and include compounds such as glycolide-co-lactide polymers.
The nano-substrate can be formed by techniques well known in the manufacture of structures of nanometer dimensions, such as those used in fabrication of semi conductor microcircuits and/or in the production of compact discs. Typically, the process employs lithography followed by etching to produce the nanometric topography, either directly into the material or so as to form a master for patterning softer materials by embossing, casting or moulding. Such techniques are described in more detail hereafter. Materials suitable for direct patterning include hard materials such as silicon, silica and perspex. Generally, materials to be produced by mechanical transfer (e.g. embossing, casting or moulding) are polymeric materials, which may be optionally heated.
The invention also relates to a master for mechanical transfer of projections or pits onto a substrate.